Circular RNA circBNC2 inhibits epithelial cell G2-M arrest to prevent fibrotic maladaptive repair

The mechanisms underlying fibrogenic responses after injury are not well understood. Epithelial cell cycle arrest in G2/M after injury is a key checkpoint for determining wound-healing leading to either normal cell proliferation or fibrosis. Here, we identify a kidney- and liver-enriched circular RNA, circBNC2, which is abundantly expressed in normal renal tubular cells and hepatocytes but significantly downregulated after acute ischemic or toxic insult. Loss of circBNC2 is at least partially mediated by upregulation of DHX9. Gain- and loss-of-function studies, both in vitro and in vivo, demonstrate that circBNC2 acts as a negative regulator of cell G2/M arrest by encoding a protein that promotes formation of CDK1/cyclin B1 complexes. Restoring circBNC2 in experimentally-induced male mouse models of fibrotic kidney and liver, decreases G2/M arrested cell numbers with secretion of fibrotic factors, thereby mitigating extracellular matrix deposition and fibrosis. Decreased expression of circBNC2 and increased G2/M arrest of epithelial cells are recapitulated in human ischemic reperfusion injury (IRI)-induced chronic kidney disease and inflammation-induced liver fibrosis, highlighting the clinical relevance. These findings suggest that restoring circBNC2 might represent a potential strategy for therapeutic intervention in epithelial organ fibrosis.


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Sample sizes for each experiment are described in figure legends. Sample sizes are determined empirically, and similar in size to most published studies in the same field (such as Nat Commun. 2022 Jan 21;13(1):438.). For in vitro assays, n=3. For animal experiments, usually n=5-8 mice were used.
Generally no data were excluded, except in the animal experiments when 1-2 mice were dropped out for analyses due to accidental deaths of the mice.
Experiments were repeated with the same conditions and obtained similar results. The number of repeats were indicated in figure legends.
Mice were randomly allocated among groups. For in vitro studies, the experiment was carried out strictly in accordance with the single variable principle. Treatment groups were divided randomly and equally.
The investigators were not blinded for the in vitro experiments because the in vitro experiments were performed by several investigators, each of them was in charge for one specific experiment from sample preparation to data analysis, therefore, the investigators could not be blinded, because they had to know the treatment for each group. The fibrosis index of mice was scored by an experienced pathologist who was blinded to the grouping. All cell lines were obtained from a trusted source and used at low passage. These cell lines were authenticated by the supplier using STR analysis.
All cells were regularly tested for mycoplasma infection and were negative.
No commonly misidentified lines were used.
Male C57BL/6, aged 6 to 8 weeks (20-24 g) were used. Mice were housed in a standard environment which was characterized by 12 h light/dark cycle, 22-25°C and 40-60% humidity with free access to water and forage. Animal welfare was reviewed during the application of the animal experiments by the Nanfang Hospital Animal Care Committee, and was monitored during the experiments by the employees of the Laboratory Animal Center of Nanfang Hospital. To euthanatize the mice before collecting tissue and blood samples, the mice were placed in a plexiglass chamber with 5% isoflurane (RWD, Cat #R510-22, Shenzhen) for 5 min. After that, cervical dislocation was performed when mice were fully sedated, as measured by a lack of active paw reflex.
No wild animals were used in the study.
No field-collected animals were used in the study.
All animal studies were approved by the Nanfang Hospital Animal Care Committee.
Human renal samples were obtained from renal biopsy specimens from Nanfang Hospital (n=12 patients with IRI-induced renal fibrosis and n=4 patients with AA-induced renal fibrosis). Tissue adjacent to clear cell renal cell carcinoma were collected as the normal controls (n=10). Liver biopsies of adult HBV-induced liver fibrosis patients (n=10) were collected from Nanfang Hospital, Southern Medical University. Normal kidney or liver tissues were obtained from 10 patients undergoing tumorectomy. Tissue adjacent to tumor was collected as the normal controls. The study was approved by the Ethics Community of Nanfang hospital. All of the study participants provided written informed consent at the time of biopsy.
The 12 patients with IRI-induced renal fibrosis includes 5 male and 7 female patients in the 29-to-52 age range at the time of renal biopsy. The 4 patients with AA-induced renal fibrosis includes 3 male and 1 female patients in the 52-to-68 age range at the time of renal biopsy. The 10 patients with HBV-induced liver fibrosis includes 6 male and 4 female patients in the 38to-53 age range at the time of liver biopsy.
The human tissue are obtained from biopsy samples. All the patients were provided with written informed consent and agreed that their tissue samples could be used for medical research.
The study was approved by the Ethics Community of Nanfang hospital. n/a